Refining of proteolytic enzymes



United States Patent "ice REFINING OF PROTEOLYTIC ENZYMES Kenneth Oringer, Spring Valley, N.Y., assignor to American Cyanamid Company, New York, N.Y., a corporation of Maine No Drawing. Application July 19, 1956 Serial No. 598,744

2 Claims. (Cl. 195- 66) This invention relates to the refining of proteolytic enzymes and more particularly is concerned with an improved method of recovering the proteolytic enzyme obtained by propagation of fungi of the order Entomophthorales upon suitable culture media.

In the copending application of Whitehill et al. Serial No. 464,642, filed October 25, 1954, there is described and claimed a method for producing a proteolytic enzyme by propagation of fungi of the order Entomophthorales in a suitable fermentation medium. The enzyme so produced has been found to be highly useful in a wide variety of industrial applications. For example, it is useful in the leather industry to prepart leather from 'raw hides, functioning as an aid in removing the epidermis and subcutaneous tissue from the raw hides. The enzyme has been found to be especially useful in the production of cheese where it functions in a manner similar to rennet in coagulating the milk but has properties superior to rennet which result in higher yields of firmer cheese curds from the milk.

As described in detail in the aforesaid copending application, the novel proteolytic substance is obtained by growing under aerobic conditions, a fungus of the species of the order Entomophthorales in an aqueous medium having a pH between 5.0 and 9.0 containing a source of assimilable carbohydrate, a source of assimilable nitrogen and traces of inorganic salts. The fungus is propagated in the nutrient medium at a temperature within the range of 20 C. to 30 C. for a period of time of about 48 to 96 hours, whereby the nutrient medium is fermented and the proteolytic enzyme is produced. The enzyme may be recovered from the fermentation broth by various means including adsorption upon diatomaceous earth and elution therefrom at a pH of from about 9.0 to 11.5.

The protcolytic enzyme so obtained is an amorphous powder having a molecular weight of about 30,000, an isoelectric point of 10.2, exerting optimal proteolytic activity at a pH of about 9.0 when measured against casein; having a sedimentation constant of 2.5Xl a difiusion constant of 82x10, an aqueous solution of ionic strength 0.13 adjusted to pH 8.5 in barbital buffer exhibiting an electrophoretic mobility of 0.43X

10- a solution of similar ionic strength adjusted to pH 10.5 in glycine buffer exhibiting an electrophoretic mobility of 0.08 the enzyme being stable over a pH range from about 4.0 to about 11.0 and giving a positive precipitin reaction against a 1:1,000 dilution of specific rabbit antiserum.

The enzyme may be produced by fermentation of a wide variety of phycomycetous fungi of the order Entomophthorales. This order is described by Bessey (Morphology and Taxonomy of Fungi, pages 172-177, Blakiston Company, Philadelphia, 1950). The order comprises a single family, the Entomophthoraceae which includes six genera: Entomophthora, Basidiobolus, Conidiobolus, Completoria, Massaspora, and Ancylistes. Al-

Patented Mar. 1, 1960 though the described proteolytic enzyme may be produced by any member of the order, it has been found that the fungi Entomophthora apiculata, Conidiobolus brefeldianus and Ewtomophthora coronata are most suitable because of the high yields of enzyme obtained.

One of the preferred procedures described in the aforesaid. application for isolating the enzyme from the fermentation medium utilizes a tannic acid precipitation of the enzyme. In this process the fermentation mash is filtered, tannic acid and sodium bisulfite are added to precipitate the enzyme, and the enzyme complex is filtered off. The complex is dissociated by treating with acetone which dissolves the tannic acid and leaves the enzyme undissolved. The enzyme is filtered off and extracted with aqueous salt solution. The enzyme is precipitated from the extract and is thereafter filtered and dried.

The tannic acid process, while producing an enzyme product of satisfactory purity, leaves a great deal to be desired from the standpoint of yields. Thus, the yields from the described process average about 40% from the fermentation mash to the final product. While this may be satisfactory on small scale runs, obviously the yield is much too low for large scale, economic productio methods.

The present invention solves this problem of yield by providing a continuous or semi-continuous process for recovering high, economically practicable yields of relatively pure protease from the fermentation mash. Thus, in accordance with the process about to be described, the yields may run as high as 90% from fermentation mash to final product with an average yield on the order of Surprisingly, in accordance with the present invention I have found that when the fermentation mash is filtered, the filtrate may be vacuum concentrated directly to the desired volume or potency. By my im proved process the number of steps necessary to isolate and purify the resulting enzyme are not only considerably reduced but the overall yields are greatly increased.

It is a surprising feature of the present invention that it has been found possible to vacuum concentrate an aqueous liquor containing a heat sensitive protease with out destroying the proteolytic activity. It has generally been considered because of the known heat-sensitivity of proteolytic enzymes that concentration of an aqueous liquor in order to increase the potency could not satisfactorily be carried out because the proteolytic activity would be destroyed as well. This has not been found to be the case, however, when the vacuum concentration of the protease obtained by propagation of fungi of the order Entomophthorales is carried out in accordance with my invention.

The vacuum concentration is carried out in suitable standard equipment for this purpose. It is preferred to concentrate the aqueous filtrate containing the activity to from 10 to 30 fold and preferably about one-tenth of the starting volume. The upper practical limit of concentration is dictated primarily by the viscosity of the mash filtrate which, when concentrated. much above about one-thirtieth of the original mash volume, results in a too viscous material from which the activity is difficult to obtain.

The desired vacuum concentration may also be expressed in terms of potency. For example, the ordinary mashes have a potency of about 300,000 Azocoll units per milliliter. With such mash filtrates I prefer to concentrate to from about three to about six million or possibly as high as nine million Azocoll units per milliliter and which represents a 10-30 fold concentration as ex! pressed above. 7

from about 60 C. until a potency of about three 1 million to about nine million Az'ocoll units per milliliter of proteolytic activity is obtained in the concenume in a natural recirculation evaporator. The results obtained are tabulated below:

Vac, Time, Cone. No. Percent Temp.,G In. Hrs. Ratio of Avg. Step Yield, 5

HQ Runs Range Example 7 The activity was precipitated from the concentrate on starch with acetone (3 vols. acetonezl vol. concentrate). 15 The precipitation was carried out at several levels of corn syrup-to-starch (0, 10, 20, v./w). The results obtained are tabulated below:

Acetone Percent Step Yield 20 Pptn Drying No. of Process Corn Method Runs yr p! Avg. Range Starch SOP"; 0.60 FD 7 00.0 77.3-07.1 25 Invert 0.20 VG 4 89.2 sac-100.7 Invert 0. 10 VC or AD 3 94.1 80. 3-1053 Invert 0.00 VCorAD.-- 8 92.8 79.7-113.

trate, precipitating the activity on a starch carrier by mixing the concentrate with acetone, and thereafter drying the precipitated product.

2. A method according to claim 1 in which the concentrate is spray dried.

References Cited in the file of this patent UNITED STATES PATENTS OTHER REFERENCES Chemistry and Technology of Enzymes, by H. Tauber, 1949, publ. by John Wiley & Sons, Inc., New York, pp. 136, 137, and 161.

American Journal of Botany, vol. 16, pp. 93-95 and 

1. A METHOD OF REFINING THE PROTEOLYTIC ENZYME OBTAINED BY AEROBICALLY GROWING A FUNGUS OF THE ORDER ENTOMOPHTHORALES SELECTED FROM THE GROUP CONSISTING OF ENTOMORPHTHORA APICULATE, CONIDIOBOLUS BREFELDIANUS AND ENTOMOPHTHORA CORONATA IN AN AQUEOUS NUTRIENT MEDIUM WHICH COMPRISES FILTERING THE MEDIUM, VACUUM CONCENTRATING THE AQUEOUS FILTRATE AT A TEMPERATURE OF FROM ABOUT 0*-60*C. UNTIL A POTENCY OF ABOUT THREE MILLION TO ABOUT NINE MILLION AZOCOLL UNITS PER MILLILITER OF PROTEOLYTIC ACTIVITY IS OBTAINED IN THE CONCENTRATE, PRECIPITATING THE ACTIVITY ON A STARCH CARRIER BY MIXING THE CONCENTRATE WITH ACETONE, AND THEREAFTER DRYING THE PRECIPATED PRODUCT. 